The objective of this study was to assess the potential utilization of melaleuca bark for Pycnoporus sanguineus mushroom cultivation. The experiment was carried out in a complete randomized design with 9 treatments (difference in substrate of melaleuca bark replacing rubber sawdust), 3 replications (10 bag/each replication). Results showed that at the first phase of culture (spawn production), mycelium got the fastest develop speed (1.78 cm/day) in the Potato-dextrose-agar (PDA) medium supplemented with 10% coconut water. In the second phase, steamed rice grain was evaluated as the optimal substrate for mycelial growth (0.988 cm/day). In the third phase, boiled cassava stalks was the best medium for mycelial spreading (0.538 cm/day). The compost consisting of combination of 60% melaleuca bark and 40% rubber sawdust was revealed as the most suitable substrate for Pycnoporus sanguineus growth that giving the highest yield (60.1 g/bag) with biological efficient (20%). In conclusion, melaleuca bark have the potential to be utilize as alternate substrate for Pycnoporus sanguineus mushroom cultivation to achieve high efficiency on the compost consisting of combination of melaleuca bark (60%) and rubber sawdust (40%) without nutritional supplement.